RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN BREAD WHEAT

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1 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN BREAD WHEAT M. R. I. ALBAKRY 1, AZIZA M. HASSANEIN 2, AMAL H. SELIM 2 1. Plant Res. Dept., Nuclear Res. Center, Atomic Energy Authority, Inshas, Egypt 2. Seed Tech. Dept., ARC, Giza, Egypt P lants have various morphological and physiological characteristics that enable them to grow and reproduce in lowrainfall environments (Johnson et al. 1983). Glaucousness has been previously reported as an adaptive trait for drought tolerance in bread wheat (Johnson et al. 1983; Fischer and Wood, 1979), in durum wheat (Clarke et al., 1994), in barley (Baenziger et al., 1983), in oat (Bengtson, et al., 1978), and in sorghum (Jordan et al., 1983). An important function of leaf epicuticular waxes (glaucousness) is to increase the efficiency of stomatal control of water loss by reducing the cuticular conductance to water vapor (Jordan et al., 1984). This advantage of glaucousness may permit more efficient use of soil water in dryland situations, and supports the finding that the ratio of net carbon exchange to transpiration is higher for glaucous genotypes (Richards et al., 1986). The inheritance of glaucousness in wheat has been studied on the conventional level (AlBakry, 9). Chromosomes 2B, 2D, and 3A were found to bear three different glaucousness genes in wheat (Stuckey, 1972). These three genes are responsible for the production of glaucousness of leaf, spike and peduncle. Molecular biology techniques in plant include genetic identification or fingerprinting of molecular markers. The technique of analyzing molecular markers is based on the detection of the DNA sequences or combinations that are unique to the individual plant under study (Henry, 1997). Molecular markers are powerful tools that can be used for markerassisted selection (Horvath et al., 1995) and as landmarks for mapbased cloning of resistance genes (Michelmore, 1995; Bai et al., 1999; Liu et al., 1). In wheat, RFLPs, AFLPs, SSRs, and RAPD markers have been used to study wheat resistance genes (Liu et al., 1; AbdelTawab et al., 3). Thus, molecular marker technology may provide new tools for the investigation of molecular markers linked to glaucousness in wheat. RAPD have been proposed by several groups as efficient tools for identification of DNA markers associated with agronomically important traits (Banerjee et al., 1999). Linkage between a polymorphic marker and the target locus is confirmed and quantified by using the segregating population from which the bulks Egypt. J. Genet. Cytol., 39: 2940, Jan., 2010

2 30 M. R. I. ALBAKRY et al. were generated. Probes or primers for loci that are polymorphic and absolutely linked to the gene or region used to distinguish the individuals comprising the bulks will detect clear differences between the bulks. Bulked segregant analysis does not reveal novel types of variation but rather allows the rapid screening of many loci and therefore the identification of segregating markers in the target region (Michelmore et al., 1991). The present study involved glaucous wheat mutant line (GWM6), nonglaucous wheat cultivar Giza 164, and three F 2 bulks resulting from the crossing between them. Bulk 1 contains glaucous type of F 2 plants; bulk 2 contains moderately glaucous type of F 2 plants; and bulk 3 contains nonglaucous type of F 2 plants. This study was conducted to identify some RAPD markers linked to glaucousness using the bulked segregant analysis (BSA). MATERIALS AND METHODS Plant materials The present study was carried out at Plant Res., Dept., Nuclear Res., Center, Atomic Energy Authority, Anshas, Egypt; and Seed Technology Dept., Agricultural Res., Center, Giza, Egypt Mapping population: GWM6 X Giza 164, developed by crossing glaucous wheat mutant line 6 (GWM 6) to nonglaucous wheat cultivar (Giza 164) was used for the genetic analysis of glaucousness on leaf blade and spike of wheat plants (Al Bakry, 9). GWM6 was developed by wheat breeding program at Atomic Energy Authority, Egypt (AlBakry, 4). It is characterized by has high epicuticular wax content on leaf sheath, leaf blade, peduncle and spike. Giza164 wheat cultivar was obtained from Wheat Dept., Crop Res., Institute, Agric. Res., Center, Egypt. Leaf blades and spikes of Giza164 cultivar are totaly nonglaucous. F 1 plants of this cross between GWM 6 and Giza 164 was moderately glaucous for both leaf blade and spike. The ratio of segregated wheat plants in F 2 was, 1 glaucous: 2 moderately glaucous: 1 nonglaucous. The occurrence of glaucous plants for leaf blade but nonglaucous for spike in F 2 segregated plants might refer to the separate inheritance of these two traits. To identify the DNA markers that are linked to glaucousness, the bulked segregant analysis (BSA) was used. Three bulks were constructed from the F 2 mapping population derived from the previous cross. Bulk 1 contains 10 F 2 plants of glaucous type; bulk 2 contains 10 F 2 plants of moderately glaucous type; and bulk 3 contains 10 F 2 plants of the nonglaucous type for both leaf blade and spike. Molecular markers Genomic DNA extraction DNeasy plant minikit (Quigen Inc., Cat.no.69104, USA) was used for DNA extraction from the GWM6 glaucous wheat mutant and nonglaucous wheat cultivar (Giza164) and the three bulked F 2 segregating populations.

3 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN WHEAT 31 RAPD PCR analysis Forty oligonucleotide primers were screened for polymorphism, and among them, eleven primers showed clear polymorphic patterns. These primers with the 5 3 sequences are shown in Table (1). The reaction conditions were optimized and mixtures were prepared (30 µl total volume) consisting of the following, DNTPs 2.4 µl, MgCl µl, 10 x buffer 3.0 µl, Primer (10 um ) 2.0 µl, Taq (5 u/µl ) 0.2 µl, Template DNA (50 ng/µl )2.0 ul, H 2 O (dd) 17.4 ul. Amplification was carried out in a PTC thermal cycler (MJ Research, Watertown, USA) programmed as follows: Denaturation, 94 C for 2 minutes, followed 40 cycles. Each cycle consisted of 1 minute at 94 C, 1 minute at 37 C, 2 minutes and 30 second at 72 C, followed by a final extension time of 12 minutes at 72 C and 4 C (infinitive). Gel electrophoresis Gel electrophoresis was applied according to Sambrook et al. (1989). Agarose (1.2%) was used for resolving the PCR products. The run was performed for one hour at 80 volt in pharmacia submarine (20 x 20 cm). Bands were detected on UVtransilluminator and photographed by Gel documentation 0, BioRad. Data analysis Bands were detected on UV transilluminator and photographed by Gel documentation 0, Bio Rad. The similarity matrices were done using gel works 1D advanced software UVPEngland program. The relationships among the parents and the three bulks under study as revealed by dendrogram were done using SPSS windows (version10) program. RESULTS AND DISCUSSION The two parents used in the present study have sufficient genetic variation regarding induced glaucousness trait. The bulked segregant analysis provides a rapid and technically simple alternative for identifying markers linked to specific genes; and the only prerequisite is the existence of a population resulting from a cross that segregates for the gene of interest. In addition, the success of this approach depends on the genetic divergence between the parents in the target region (Michelmore et al., 1991). Thus, the mapping population resulting from the crossing between these two contrasting parents under study is suitable for identifying molecular markers associated with induced glaucousness. Polymorphism detected by RAPD analysis In the present study, RAPD analysis was used to determine the genetic variation and molecular markers associated with induced glaucousness genes. Eleven arbitrary primers were used for PCR amplification of the genomic DNAs of GWM6 (P 1 ), Giza 164 (P 2 ), F 2 glaucous bulk, F 2 moderately bulk and F 2 nonglaucous bulk of the wheat cross GWM6 x Giza164. The total number of amplified

4 32 M. R. I. ALBAKRY et al. fragments produced by the 11 arbitrary primers is presented in Table (2). Among the 11 arbitrary primers, amplification products of five primers (45.45%) generated polymorphic RAPD markers linked to induced glaucousness (Fig. 1). However, amplification products of six primers (55.55%) generated monomorphic RAPD fragments (Fig. 2). The number of amplified fragments produced per primer varied from 10 (OPB14, OPC06, OPC11 and 10) to 46 (OPB03). A total of 51 DNA amplicons, ranging from to 2, were generated for the tested genotypes (Table 3). Out of these amplified DNA amplicons, 34 were conserved (monomorphic) among all genotypes, while 17 were polymorphic. The number of RAPD amplicons produced by each primer varied from 2 (primers 0PB14, 0PC06, 0PC11, 0PC20) to 11 (primer 0PB03) with an average of 5 per primer. The highest numbers of polymorphic DNA fragments were 5 (0PB13), while the lowest number was 0 (primers 0PA10, 0PB14, 0PC06, 0P C11 and 0PC20). The total number of monomorphic amplicons was 34. The number of polymorphic amplicons was 17. The number of amplified polymorphic DNA amplicons across the 5 primers ranged from 1 to 5 amplicons. The maximum number of amplicons was amplified with the primer 0P B03, while the primer 0PA09 amplified the minimum number. However, the percentage of polymorphism ranged from 25%was shown by 0PA09 and 0PC08 primers to 71% shown by 0PB13 primer. Genetic relationship among genotypes studied Bands generated from the 11 RAPD primers, were utilized to calculate the genetic similarity index (RAPDGS) among GWM6, Giza164 cultivar and the three bulked F 2 segregated populations (Table 4). Genetic similarity (GD) among wheat genotypes ranged from 0.82 to 0.95, with an average of The smallest genetic similarity (82%) was observed between glaucous parent and nonglaucous F 2 bulk, while the largest similarity (95%) was between glaucous parent and glaucous F 2 bulk followed by 93% between nonglaucous parent and nonglaucous F 2 bulk. The dendrogram based on cluster analysis of RAPD data is presented in Fig.. (3). The genotypes under study were separated into two distinct groups, the first group is separated into two subgroups the first subgroup included glaucous parent and glaucous F 2 bulk, while, the moderately glaucous was in a separate subgroup. The second group was composed of nonglaucous parent and nonglaucous F 2 bulk. RAPD markers associated with induced glaucousness Out of the eleven primer pairs tested in this study, five primers gave polymorphism and developed molecular markers associated with glaucousness (Table 5). These molecular markers were based on polymorphism between the DNA of glaucous and nonglaucous genotypes.

5 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN WHEAT 33 Two molecular markers, appeared in the glaucous parent and glaucous F 2 bulk, were detected by the primers 0PB03 and 0PB13 at molecular sizes of 527 and 1312, respectively, while there were two bands with molecular sizes of 1453, 456 of primer 0PC08. On the other hand, three molecular markers were detected in the glaucous parent, glaucous F 2 bulk and moderately glaucous F 2 bulk. These three markers were detected by primers 0PB13, 0PB15 and 0PC05, respectively, at 634, 888, 622. In wheat no molecular markers associated with glaucousness related genes were reported before in a study conducted by Liu et al. (7). They investigated the genetic control of nonglaucousness in synthetic Line 3672 and to map the gene(s) using molecular markers. They found that nonglaucousness in that Line was controlled by a single dominant gene, temporarily designated Iw3672. Five SSR markers linked to Iw3672 gene were mapped on chromosome 2DS. In addition, two ESTderived markers and a SNP marker were developed and were also linked to Iw3672 gene. The conventional plant breeding is primarily based on the selection of superior individuals among segregating progenies of sexual matings. In addition, selection for plant improvement has largely been carried out on the wholeplant or phenotype, which is the result of genotype and environmental effects. Thus, conventional plant breeding is often hampered by difficulties in selecting for agronomically important traits, especially when they are considerably influenced by the environment. Moreover, testing procedures may be difficult, unreliable, or expensive, due to the nature of the target traits or the target environment. For these reasons, selection through molecular markers might be an efficient complementary breeding tool, especially when selection is done under unfavorable conditions (Ribaut et al., 1). The associated molecular markers with induced glaucousness identified in the present study can greatly enhance the efficiency of incorporating glaucousness into new varieties. This favorable allele can be introgressed in new target germplasm through backcrossing, or can be selected in a segregated population i.e., the seven RAPD markers associated with induced glaucousness detected in the present study can be used in markerassisted selection. SUMMARY The present study was conducted to identify some DNA markers that are linked to glaucousness. RAPD analysis was used to determine the genetic variation and molecular markers linked to induced glaucousness genes. Eleven arbitrary primers were used for PCR amplification of the genomic DNAs of GWM6 (P1), Giza 164 (P2), F 2 glaucous bulk, F 2 moderately bulk and F 2 nonglaucous bulk of the wheat cross GWM6 x Giza164. Among the 11 arbitrary primers, amplification products of 5 primers (45.45%) generated polymorphic RAPD markers linked to glaucousness. However, amplifi

6 34 M. R. I. ALBAKRY et al. cation products of 6 primers generated monomorphic RAPD bands. The number of amplified bands produced per primer varied from 10 to 46. A total of 51 DNA amplicons, ranging from to 2, were generated for the tested genotypes. Out of these amplified DNA amplicons, 34 were common (monomorphic) among all genotypes, while 17 were polymorphic. The number of RAPD amplicons produced by each primer varied from 2 to 11 with an average of 5 per primer. Out of the eleven primer pairs tested in this study, five primers gave polymorphism and developed molecular markers associated with glaucousness. Two molecular markers, appeared in the glaucous parent and glaucous F 2 bulk, were detected by the primers 0PB03 and 0PB13 at molecular sizes of 527 and 1312 respectively, while two molecular markers were observed with molecular sizes of 1453 and 456 of primer 0PC08. On the other hand, three molecular markers were exhibited in the glaucous parent, glaucous F 2 bulk and moderately glaucous F 2 bulk by primers 0PB13, 0PB15 and 0PC05, respectively, at 634, 888 and 622. REFERENCES AbdelTawab, F. M., Eman M. Fahmy, A. Bahieldin, Asmahan A. Mahmoud, H. T. Mahfouz, Hala F. Eissa and O. Mosellhy (3). Markerassisted selection for drought tolerance in Egyption bread wheat. Egypt. J. Genet. Cytol., 32: AlBakry, M. R. I. (4). Improvement of wheat for drought tolerance by using some biotechnological and nuclear techniques. Ph. D. Thesis, Fac. Agric. Cairo University, Egypt. AlBakry, M. R. I. (2010). Inheritance of induced glaucousness, grain yield, and yieldrelated traits in a cross between glaucous mutant line and nonglaucous cultivar of bread wheat, Triticum aestivum L. Egypt. J. Genet. Cytol., 39: Baenziger, P. S., D. M. Wesenberg and R. C. Sicher (1983). The effects of genes controlling barley leaf and sheath waxes on agronomic performance in irrigated and dry environments. Crop Sci., 23: Banerjee, N. S., P. Manoj and M. R. Das (1999). Male sex associated RAPD markers in Piper longum L. Curr. Sci., 77: Clarke, J. M., T. N. McCaig and R. M. DePauw (1994). Inheritance of glaucousness and epicuticular wax in durum wheat. Crop Sci., 34, Fischer, R. A. and J. T. Wood (1979). Drought resistance in spring wheat cultivars. III. Yield associations with morphophysiological traits. Aust. J. Agric. Res., 30:

7 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN WHEAT 35 Henry, R. J. (1997). Practical Applications of Plant Molecular Biology. IstEdn., Published Chapman and Hall, Chapman, ISBN: Horvath, D. P., L. S. Dahleen, J. A. Stebbing and G. Penner (1995). A codominant PCRbased marker for assisted selection of durable stem rust resistance in barley. Crop Sci., 35: Jordan, W. R., R. L. Monk, F. R. Miller, D. T. Rosenow, L. E. Clark and, P. J. Shouse (1983). Environmental physiology of sorghum. I. Environmental and genetic control of epicuticular wax load. Crop Sci., 23: Liu, X. M., C. M. Smith, B. S. Gill and V. Tolmay (1). Microsatellite markers linked to six Russian wheat aphid resistance genes in wheat. Theor. Appl. Genet., 102: Michelmore, R. W., I., Paran and R. V. Kesseli (1991). Identification of markers linked to disease resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA, 88: Michelmore, R. W. (1995). Isolation of disease resistance genes from crop plants. Curr. Opin. Biol., 6: Pakniyat, H and E. Tavakol (7). RAPD markers associated with drought tolerance in bread wheat. Pakistan Journal of Biological Sciences 10(18): Ribaut, J. M, H. M. William, M. Khairallah, A. J. Worland and D. Hoisington (1). Genetic Basis of Physiological Traits. In, Application of Physiology in Wheat Breeding. Reynolds, M.P., J.I. Ortiz Monasterio, and A. McNab (eds.). Mexico, D.F.: CIMMYT. Richards, R. A., H. M. Rawon and D. A. Johnson (1986). Glaucousness in wheat: Its development and effect on wateruse efficiency, gas exchange and photosynthetic tissue temperatures. Aust. J. Plant Physiol., 13: Sambrook, J., E. F. Fritsch and T. Maniatis (1989). Molecular cloning. A Laboratory manual, second edition. Volume 1. Stuckey, J. R. (1972). Inheritance of glaucousness in wheat. Ph. D. Thesis, Univ. of New South Wales, Sydney, Australia.

8 36 M. R. I. ALBAKRY et al. Table (1): Random primer names and their sequences used for RAPD PCR analysis. Primer name Sequence Primer name Sequence 0PA09 5 GGGTAACGCC 3 0PC05 5 GATGACCGCC 3 0PA10 5 GTGATCGCAG 3 0PC06 5 GAACGGACTC 3 0PB03 5 CAT CCCCCTG 3 0PC08 5 TGGACCGGTG 3 0PB13 5 TTCCCCCGCT 3 0PC11 5 AAAGCTGCGG 3 0PB14 5 TCCGCTCTGG 3 0PC20 5 ACTTCGCCAC 3 0PB15 5 GGAGGGTGTT 3 Table (2): Number of amplified DNA fragments scored for GWM6, Giza164, F 2 glaucous bulk, F 2 moderately bulk and F 2 nonglaucous bulk of the wheat cross GWM 6 x Giza 164. Primer GWM 6 Number of amplified DNA bands Giza16 4 F 2 glaucous bulk F 2 moderately bulk F 2 nonglaucous bulk Total Mean OPA OPA OPB OPB OPB OPB OPC OPC OPC OPC OPC Total Table (3): Levels of polymorphism based on RAPD analysis scored for GWM6, Giza164, F 2 glaucous bulk, F 2 moderately bulk and F 2 nonglaucous bulk of the wheat cross GWM6 x Giza164. Primer Number of Polymorphic Monomorphic Polymorphism amplicons amplicons amplicons % 0PA % 0PA % 0PB % 0PB % 0PB % 0PB % 0PC % 0PC % 0PC % 0PC % 0PC % Total

9 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN WHEAT 37 Table (4): RAPDbased genetic similarity (GS) matrices among the GWM6 (P 1 ), Giza164 (P 2 ), F 2 glaucous bulk, F 2 moderately glaucous bulk, and F 2 nonglaucous bulk of the wheat cross GWM6 x Giza164 based on RAPD analysis. Genotype GWM6 Giza164 F 2 glaucous bulk F 2 moderately glaucous bulk Giza F 2 glaucous bulk F 2 moderately glaucous bulk F 2 nonglaucous bulk Table (5): RAPD markers and their molecular sizes () associated with induced glaucousness based on DNA of GWM6, F 2 glaucous bulk and F 2 moderately glaucous bulk of the wheat cross GWM6 x Giza164. Genotype GWM 6 F 2 glaucous bulk F 2 moderately bulk Marker size () Primer 0PB03 0PB13 0PB13 0PB15 0PC05 0PC08 0PC08 0PB03 0PB13 0PB13 0PB15 0PC05 0PC08 0PC08 0PB13 0PB15 0PC05 Total 7 7 3

10 38 M. R. I. ALBAKRY et al OPB03 OPB OPB15 OPC05 Fig. (1): RAPD banding patterns for 1:GWM6 (P 1 ), 2: Giza164 (P 2 ), 3: F 2 glaucous bulk (G), 4: F 2 moderately glaucous bulk (MG), and 5: F 2 nonglaucous bulk (NG) M: DNA Marker (+1.5 kb ladder marker) OPC08

11 RAPD MARKERS LINKED TO INDUCED GLAUCOUSNESS IN WHEAT OPA09 OPA OPB14 OPC OPC11 OPC20 Fig. (2): RAPD banding patterns for 1:GWM6 (P 1 ), 2:Giza 164 (P 2 ), 3:F 2 glaucous bulk (G), 4:F 2 moderately glaucous bulk (MG), and 5:F 2 nonglaucous bulk (NG) M: DNA Marker (+1.5 kb ladder marker).

12 40 M. R. I. ALBAKRY et al. Fig. (3): Dendrogram based on cluster analysis of RAPD data. 1:GWM6 (P 1 ), 2:Giza 164 (P 2 ), 3:F 2 glaucous bulk (G), 4:F 2 moderately glaucous bulk (MG), and 5:F 2 nonglaucous bulk (NG)